378 Lect Notes 13

Light, Optics and the Microscope
I. II. Why we should consider these subjects: General outline of the subject: A. Description of light B. The electromagnetic spectrum C. Refraction, refractive index D. Lenses E. Simple and compound microscopes F. Aberrations and their corrections G. Resolution and magnification Light (slides 1-3, 1-4): A. Electric field B. Magnetic field C. Direction of propagation D. Speed of propagation. = 3 x 10^8 m/sec in vacuum E. The simplified representation: Usually a light ray is drawn in a simplified manner. In this format only the electric component is shown in a planar waveform. The height of the waves is an indication of the intensity of the light ray. F. Wavelength: Varies over a wide range, only a few of which are normally seen as "visible light". I have listed below a number of parts of the electromagnetic spectrum and their respective wavelength. (You do not have to memorize this.) type of radiation gamma rays x-rays U.V. light Violet Blue Green yellow red infrared radio waves G. wavelength 10-4 to 10-2 nm 10-2 to 10 nm 10-400 nm 400-450 nm 450-500 nm 500-560 nm 560-600 nm 600-700 nm 700 nm to 1 mm up to km
III.
Interference: When two light waves of the same polarity are traveling in the same direction, their amplitudes can be added together at each point to obtain a resultant wave. If the two light waves in question have the same wavelength, this addition could result in constructive or destructive interference. 1. Constructive interference (slide 1-5): If the two or more waves are in phase, than the resultant wave has an amplitude that is the sum of the original waves. Because amplitude of the wave is an indicator of energy, this wave would look bright. 2. Destructive interference (slide 1-6): If two or more waves are exactly out of phase, the resultant wave will be the difference between the amplitudes of the original waves. If the two original waves were nearly the same amplitude, the resultant wave will have a low amplitude and thus would not look bright.
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Microscope Techniques
Partial destructive interference: If two waves are partially out of phase, the resultant wave will have an amplitude lower than that of the wave produced by constructive interference, but higher than that produced by destructive interference. The amplitude of the resulting wave is determined by the relative phase shift; rays nearly in phase will produce a relatively large amplitude resultant, rays nearly out of phase will produce a relatively small amplitude resultant. 4. Interference phenomenon determine the ultimate theoretical resolving power of the microscope, and are exploited in both phase contrast microscopy and differential interference microscopy. We will consider these in more detail in the next few weeks. IV. Refractive index: A. Although all electromagnetic waves travel at the same speed in vacuum, the waves are slowed down by matter (slide 1-7). The ratio of the velocity of the wave in vacuum divided by the velocity of the wave in a given substance is called the refractive index. Different substances can have markedly different refractive indices (slide 1-8). B. A beam (ray) of light bends as it moves from a substance of one refractive substance into another of a different refractive at any angle other than the normal (slide 1-9) C. Going from a substance of lower refractive index to one of higher refractive index, it will bend towards the normal. Going from a substance of higher refractive index to lower, it will bend away from the normal. This is why a fish underwater looks as if it is in a different position from where it really is. V. The lens: Now lets what happen when two parallel beams of light pass through a convex lens. A. As the rays enter the lens each ray bends to the normal of the air/glass interface. Because the surface is curved the rays are no longer traveling in parallel paths, but bend towards one another (slide 1-10). B. Further, as the rays exit the glass, they are bent away from the normal (slide 1-11), again bending towards one another. C. Thus a convex glass lens in air is a converging lens (slide 1-12). VI. Real and virtual images: A. The real image: 1. A lens or lens system can be used to form a real image that is larger or smaller than the actual object. Such an image is called real because it is located in a particular space. A translucent screen held at the right place would allow you to see the image from many angles. 2. An example of a real image: Note that the slides we have used make a real image on the screen. If the screen was translucent we would be able to see it from all sides. 3. It turns out that the objective of the compound microscope also projects a real image of the specimen much like the slide projector. B. The virtual image 1. In contrast a virtual image is an image made in the mind. Virtual images can have a perceived position, but will not project an image onto a card. 2. An example of a virtual image: when you look in a mirror, you appear to see another person on the other side of the mirror. However, the apparent position of the image may be several feet in back of a solid wall. 3. When you look into a microscope (or telescope) the enlarged image will appear to be located about a foot from the eye. However, there is no real image in that position. No one else can see that image even if you hold a translucent screen where you see the image.
Microscope Techniques Page 2
3.
VII. Simple microscopes (the magnifiers; slide 1-13): A. The eye cannot focus at very close distances, so there is a limit to the ability to magnify an object by bringing an object closer to the eye. B. However consider a converging lens placed between the object and your eye. If the distances between the object and the lens and the lens and the eye are suitable, a sharp image of the object will be projected to the eye. The mind will form a virtual image at about a foot that will be much larger than the object. C. The usable magnification of this system is rather limited. Perhaps the most impressive use of such a system was the microscopes of Leeuwenhoek. Incidentally, to use his microscopes the lens must be held right up against the eye, certainly an uncomfortable position. VIII. The compound microscope (slide 1-14): A. This is what we think of when we talk about a microscope. B. Notice that it is a two-step process: 1. First, the microscope objective makes a magnified real image of the object. 2. Then, the ocular is used to further magnify the image. 3. As we saw with the simple microscope, the mind creates a virtual magnified image about a foot from the eye. In fact, it is possible to place a piece of paper at that distance, and, while looking in the microscope with one eye, trace the outline of the image by looking at the pencil and paper with the other. IX. Aberrations and their corrections: Up to now, we have considered only "ideal" lenses. However, lenses are never really ideal. Aberrations are not the result of poor design, but are the result of basic physical principles inherent in simple lenses and lens systems. Most aberrations cannot be completely eliminated even in very high quality optics. A. Spherical aberration: 1. Cause: most lenses are composed of segments of a sphere as the result of the manufacturing process (slide 1-16). In such a lens the rays that go through the periphery of the lens will focus in front of rays that go through the center (Slide 117) 2. Possible remedies: a) Use only central portion of lens. b) Use aspherical surfaces. Generally difficult to grind lenses this way and therefore such lenses are expensive to make. Also, it is difficult to solve for aberrations so it generally works better for simple lens systems than complex ones. Aspheric lenses are sometimes used for the condenser and ocular, but I know of no microscope manufacturer who does this with regular objectives. c) Use combinations of lenses. This is the way good microscope lenses are corrected. B. Chromic (or chromatic) aberration (slide 1-17): 1. Cause: Different wavelengths are slowed to a different degree as they enter the glass, and thus light of each wavelength is bent to a different degree. This is exactly what happens in a prism.) This in turn results in different focal lengths for the different wavelengths. The blue focuses in front other colors while red focuses in back. 1. Remedies: a) Use a filter to give monochromatic light. This is often done with inexpensive microscopes. b) Combine several lenses to make a lens system with reduced chromatic aberration (see 1-18). For example, in b. a relatively weak diverging lens
with high dispersion is combined with converging lenses of greater total power, but approximately equal dispersion. C. Field curvature (slide 1-19): 1. Cause: the image plane produced by a curved lens is normally also curved. This results in only part of the field being in focus at any one time. With thin flat specimens (such as a blood smear) only a small portion of the field may be in focus at any one time. Field curvature is especially a problem in photography, where the ultimate viewer cannot use the fine focus knob to bring the edges into focus. 2. Remedy: Use of lens systems to correct for flat field. D. Other faults: The following are not inherent in the design of simple lenses, but are the result of imperfect design or manufacturing of lenses or lens systems. Therefore, strictly speaking they are not aberrations, although in common language they are frequently referred to in that way. 1. Coma: Off axis points do not look like a point but rather like a comet. 2. Astigmatism: Horizontal and vertical lines have different levels of focus. Common in the human eye and some of you may have glasses to correct for this condition. In microscope lenses, this results in a fuzzy image. 3. Distortion: Objects in the field do not display their true shape. Most common kinds are "pincushion" and "barrel" (slide 1-20). X. Magnification and resolution: A. Magnification is simply the relative size of the image (virtual or real) and the object. By using more powerful lenses or a greater distance between the objective and the ocular, an image can be made as large as you want. We could even project and image of a bacterium on the moon (assuming that we had a bright enough light). However, magnification, by itself is not very useful to a biologist. B. As a first approximation, resolution is the ability to see detail. Resolution is the reason we use a microscope. C. An example of the difference between magnification and resolution: (slide 1-21). D. We will return to resolution and factors lead that influence it after we have talked about the components of the compound microscope.
The Brightfield Microscope

I. The concept of the optical train (slides 1-23 1-27) A. Light source including bulb, collector lens, field diaphragm. B. Conditioner condenser iris, DIC prisms. phase annulus, filters, etc. C. Condenser D. Specimen E. Objective F. Image filters (DIC prism, phase ring, barrier filters, analyzer etc.) G. Ocular H. Receiver (eye, film camera, video camera, etc.)
II. Strategy for study: We will start with the objective and ocular, than work through the specimen, condenser, and light source. If you really understand the brightfield microscope, it will be relatively easily to understand the other types of light microscopy such as phase, DIC, polarizing etc. III. Objective A. Purpose: The objective magnifies the object and supplies a magnified real image to the ocular. It is here that the constraints placed on the optics are the most severe: thus it is this component that is most responsible for the resolution of the system. It is also one of the most expensive portions of the microscope, good lenses commonly cost $1,000-3,000 each! B. Objectives come in a bewildering array of types. One manufacturer may make thousands of different objectives. Objectives can differ in: 1. Magnification 2. Numerical aperture: The numerical aperture is defined as the half angle of the cone of light entering or leaving a lens system (times the refractive index of the immersion medium -- one in the case of air). Thus, the numerical aperture will generally be larger with higher magnification objectives (slide 1-29). As we shall see, the larger the numerical aperture, the higher the resolving power (assuming perfect lenses). Thus the higher the N.A. the better (and the more expensive) the lens. 3. Working distance: In general the working distance decreases as the magnification and resolution increases. However, this is not an optical law, and most manufactures also make special longer working distance condensers and objectives for use in micromanipulation, tissue culture etc. 4. Immersion medium: a) air: usually not marked or marked dry b) water c) glycerol d) oil 5. Types of chromatic and spherical aberration corrections (slide 1-30) (The following specifications are minimum. Many manufacturers correct their lenses for more wavelengths then suggested below) a) Achromats (achromatic lens: chromatic aberration corrected for 2 wavelengths, usually red and blue. The paths of the other wavelengths are presumed to be close to one or the other (but see slide 1-18!) Spherical aberration is corrected for one wavelength. b) Fluorites, Semi-apochromats: chromatic aberration corrected for 3 wavelengths. Spherical aberration is corrected for a single wavelength. Semi-apochromats often have fluorite glass in one or more optical elements. c) Apochromats: chromatic aberration corrected for at least three wavelengths and for spherical aberration corrected for 2 wavelengths. (See slide 1-31 for an example of the construction difference between an achromat and a apochromat.) 6. Presence of other correction or special attributes. a) Flat field (= plan) b) Strain free (used in DIC, polarizing) c) Modified for special contrasting methods such as phase contrast.
7. Tube length This is the distance from the back focal plane of the objective to the position of the real image that is in turn magnified by the ocular. Usually this is 160 mm, but some microscope systems are designed with other tube lengths. Recently, some manufactures are going to optics optimized for an infinite path length. The objectives cannot be used on the old microscopes (and visa-versa) without modification. C. How to identify the objectives (slide 1-32) and below: Almost all objectives have cryptic notes on the lens barrel that tell you quite a bit. Most objectives have the manufactures, magnification, power, and numerical aperture right on the barrel. Semi-apochromats, and apochromats will also usually be specified (of course! These are the expensive lenses and the manufactures want to brag). Examples of lens marking are listed below. 1. Zeiss PlanApochromat, 40x/1.0 Oil Iris, Infinity/0.17 Manufacturer: Zeiss Mag 40x Correction: apochromat Numerical aperture: up to 1.0 (depending on setting of iris) Immersion medium: oil Tube length: infinity Cover slip thickness: 0.17 mm Special attributes: flat field, iris 2. Bausch & Lomb 10x, flat field Manufacturer Bausch & Lomb Mag. 10x Correction: not specified. Hopefully it is an achromat but it may not even be corrected. Numerical Aperture: not specified Immersion medium: air Tube length: not specified, probably 160mm Special attribute: flat field 3. AO Spencer, 16 mm, NA 0.30, Apoch., 10x Manufacturer: American Optical/Spencer Mag.: 10x Correction: Apochromat Numerical Aperture: 0.30 Immersion medium: air Tube length: not specified, probably 160 mm 4. AO 20 /.50, Plan Achro, SF Manufacturer: American Optical Mag.: 20 x Correction: Achromat Numerical aperture 0.50 Immersion medium: air Tube length: not specified, probably 160 mm Special attributes: flat-field, strain free
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IV. Oculars: A. Purpose: the ocular further magnifies the real image produced by the objective to produce a magnified virtual image (when viewing through the microscope) or real image (when photographing). The optical constraints are not nearly so great as in the case of the objective, so the ocular are usually much less expensive. B. Differences in oculars: 1. Tube length: mentioned previously. Obviously, the real image projected by the objective must be in the proper place or it cannot be imaged by the ocular. 2. Final correction for chromatic aberration. Many manufactures use the ocular to correct residual lateral chromatic aberration. Oculars so designed are referred to as compensating oculars. So a microscopist can use a single set of oculars with all objective lenses, many manufacturers design the ocular to correct for the worst chromatic aberration produced by any of their objectives. They then purposely design the other objectives to add chromatic aberration to match the correction present in the oculars. 3. Obviously, it is good practice to use an ocular from the same company that you purchase the objective. C. Choice of magnification for oculars: The ocular merely magnifies the real image produced by the objective, so the ocular does not directly contribute to resolution of the microscope. However, if the oculars are of insufficient power, the final image is not large enough for the human eye to pick out all the information that is present in the image. Because people differ in their visual acuity, the minimum magnifying power needed is different for each person. If the ocular has more magnification power than needed, the extra magnification reduces the width of field without enabling the investigator to see any additional detail. For good microscopist and most objectives, a 10x magnification is sufficient. Most oculars have a magnification of between 5 and 20x. V. Specimen: One of the most overlooked parts of the optical path. For the following discussion, the specimen will be taken to be anything between the condenser and the objective and will include the object, slide, coverslip, mounting medium, and immersion medium (slide 135). A. The coverslip: 1. The effect of a coverslip on image quality (slide 1-36). 2. The reason for this effect (slide 1-37). As you can see, the coverslip affects the light path, especially of rays at the periphery. The objective lens has been designed to take this in account, therefore trying to use an objective designed for a coverslip without one will result in a poor image. Most microscope objectives are designed for a coverslip of 0.17 +/- 0.01 mm thick with refractive index of 1.515. 2. How to match coverslips with objectives: a) For critical work can measure each coverslip individually with a micrometer, otherwise use #1 1/2 coverslips which come close to the ideal. b) Some objectives have a correction collar so the microscopist can correct for each coverslip. c) Objectives can be designed for use without coverslips, but are generally expensive and special purpose. For example, blood smears are frequently examined without a coverslip, so for critical work an NC (no coverslip) objective should be used.
d) Matching the coverslip to the objective is most critical for high dry objectives. If oil is used there is much less refraction at the surface of the coverslip. Therefore, oil lenses can be used with mismatched or absent coverslips with a minimal loss of resolution. B. Oil immersion: 1. The critical angle is the angle at which light, traveling from a substance of one refractive index (usually higher) to another (usually lower), cannot pass, but is instead reflected. (slide 1-38). 2. It is important to microscopist because it limits the amount of light (and hence information) that can be gathered (or delivered) by an objective (or condenser) lens (slide 1-39). Because the ultimate resolution of the system is dependent on the numerical aperture of the objective and condenser and because the numerical aperture is dependent in large part on the refractive index of the immersion medium, oil immersion optics are used for the highest resolution possible. 3. How to use oil: a) Only use oil on objectives and condensers designed for it. Oil will not improve the optics of dry objectives because they have not been corrected for the light rays that would be introduced in the periphery of the lens. Oil can quickly ruin non-immersion lenses by getting between optical elements. b) Use only the immersion medium for which the lens is designed. The lens will not necessary be corrected for immersion media of differing refractive indexes. c) Avoid bubbles in oil d) If your condenser is designed to use oil, you should put oil between the condenser and specimen any time you use oil between the specimen and objective. e) Be careful using oils manufactured some time ago. Some of these oils were filled with PCBs, now known to be toxic. VI. The condenser: A. The importance of the condenser The condenser does three important things: 1. It focuses light so the specimen is brightly lit. This was the first use of the condenser in early microscopes. Remember that if the image is magnified 1000x, the light going through the specimen has been spread over an area 1,000,000 as great as the original specimen before viewing. 2. It conditions the light so that the light will interact optimally with the specimen. 3. It can contain portions of imaging systems for control of contrast etc. B. Considerations on the use of the condenser 1. If you are responsible for purchase of a microscope system, do not skimp on the condenser. It is nearly as important for a good image as the objectives. 2. Condensers, like objectives, come in different degrees of correction. The most highly corrected condensers are referred to as achromatic aplantic (corrected for chromatic and spherical aberrations). The best condensers also have a high numerical aperture. 3. If you are going to use immersion objectives, get and use an immersion condenser. (The only exception is when epiillumination is used where no condenser is necessary.) In biology, this type of illumination is used mainly in indirect immunofluorescence (see The fluorescent microscope)). For discerning
microscopy, make sure the condenser is matched your illuminating system and to the objectives. The brightfield condenser has a built-in condenser diaphragm, This is used to regulate the working NA of the condenser-NEVER use to control light intensity. VII. The illuminating system: This is probably the most abused part of the microscope. The microscope image can never be better than the system that supplies the light, but careful attention to the illuminating system can result in a pretty good image from a marginal scope. A. Parts of the illuminating system (slide 1-40): 1. Light source: In general the brighter the better, especially if the microscope is capable of contrast control such as phase contrast etc. 2. Lamp condenser (field condenser, field lens): designed to focus the light into the condenser. 3. Lamp iris (field diaphragm, field iris). This regulates the area of the specimen to be illuminated. It should not (and in a properly set up microscope cannot) be used to control light intensity. 4. Diffusing screen: In inexpensive microscope can be used in place of 2 and/or 3 above. B. Types of illumination systems: 1. Diffuse: a) Least expensive, does not need lamp iris or lamp condenser. b) Easy to set-up, almost no adjustments possible or needed. c) Not a very satisfactory illumination system for exacting requirements. Image is normally less bright and it is not possible to adjust illumination for utmost resolution. d) Most applicable for use in microscopes that will be used by untrained people, beginning students. 2. Koehler (Slide 1-40): a) Basic premise: The image of the lamp filament is focused onto the plane of the condenser diaphragm. In addition, the image of the field diaphragm is focused onto the specimen plane. b) Advantages: (1) The field is homogeneous and bright. (2) The working NA of the condenser and the size of the illuminated field can be manipulated separately. (3) Gives the maximum lateral resolution. (4) Gives the finest optical sectioning (longitudinal resolution) (5) Flair resulting from optics and barrels is reduced (6) Gives optimal contrast c) Disadvantages: (1) Is more difficult to set up than diffuse illumination (2) Set up incorrectly it will not give optimal results (3) Is more expensive than diffuse illumination d) How to set up Koehler: Note: This will only work if the microscope illumination system has been designed for Koehler. This must be done every time you change objectives. (1) Focus on specimen (slide 1-42). (2) Close field diaphragm.
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(3) Raise or lower condenser so you see a crisp image of the field diaphragm (slide 1-43). (4) Center the image of the field diaphragm (slide 1-44). (5) Open the field diaphragm so that the edges are just visible at the edges of the field (slide 1-45). (6) Adjust condenser diaphragm: (a) Replace the ocular with a focusing telescope and adjust telescope to visualize the condenser diaphragm. or Move Bertrand lens into position or Remove ocular and look down eyepiece tube (b) Adjust the condenser diaphragm so that it results in a lit area of about 2/3 of the total area. Now the working condenser numerical aperture is slightly less than that of the objective. (c) Replace the focusing telescope with the objective or Remove Bertrand lens or Replace ocular (7) Check image and contrast. The maximum resolution is with the working NA of the condenser equal to that of the objective, so if resolution is critical the condenser diaphragm can be opened a little. On the other hand, closing the condenser further will increase contrast. In general, the condenser diaphragm should be open as far as possible consistent with the required contrast (but never further than is required to make the NA of the condenser equal to that of the objective). (8) A warning: Note that neither the field diaphragm not the condenser diaphragm should be used to regulate light intensity, because both are adjusted for the proper optical requirements. Instead use light intensity controls or neutral density filters. VIII. How to recognize brightfield: A. As the name suggests, the surround is normally bright (white). 1. But occasionally a microscopist will place a colored filter over the light source so the background (and the specimen) would be colored. 2. Also sometimes people put ink around the nominal specimen to block out light that would otherwise make up the image. This does not break the rule, b/c the ink particles are themselves specimens, even if they are not the main point of the micrograph. B. Many biological specimens are normally near colorless, so the specimen will often be artificially stained. C. The lack of characteristic features that go with the other contrasting techniques. D. A gallery (slides 1-62 to 1-65)
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Important Optical Characteristics of the Light Microscope

I. Resolution: Perhaps the most important optical characteristic: A. Definitions: 1. Informal: Up to now we have simply referred to resolution as the ability to see detail. This is still a good working definition but is sometimes not sufficient. 2. Formal (slide 1-47 -- 1-48): The resolution of a system is the minimum distance at which two very small but bright (or dark) objects can be distinguished from one another. Note that an object smaller than the resolving power of the system can be observed but it is not possible to visually separate two or more of these objects if they are closer to one another than the limit of resolution. B. What determines resolution? 1. Wavelength of light used: a) The shorter the wavelength, the better the theoretical resolution. However the wavelength of visible light varies over a small range, so one can only make minor improvements over white light by using blue or green rather than red light. It is possible to use U.V. light for a slight increase in resolving power but one must use quartz lenses (U.V. does not penetrate most optical glass) and a special viewing system (U.V. cannot be seen by the human eye and is very bad for eyes. However, the relatively small increase in resolving power does not make up for the considerable expense and complexity necessary for such as microscope. Consequently U.V. microscopes are very rare. 2. The numerical aperture of the system: The numerical aperture of a objective lens or condenser is defined as the sine of the half cone angle of light entering or leaving the lens system times the refractive index of the immersion medium. The higher the numerical aperture of a lens system, the higher the amount of information that can be captured by the lens system and the greater the potential resolution. C. The formula to determine maximum theoretical resolution: We have talked about resolution in general terms until now. However, we finally have a chance to quantify it for a given system. The theoretical resolution (that is the smallest distance between two points that still results in those points being resolved) is: r = 1.2 (wavelength of light used) / (NA obj + NA cond). Where the NAs are working NAs; not necessarily the ones marked on the lenses. Sometimes this equation takes different forms, especially if the author is talking about self-luminous objects, or other simplified or specialized cases. D. Example: About the best one can do with a light microscope is: Wavelength = 450 nm (blue/violet) NA obj. = 1.4 NA cond. = 1.3 (1.2) (450 nm)/(1.4 + 1..3) = 200 nm = 0.2 um E. Note that this is the best resolution possible and requires perfect optics and careful microscope adjustment. Fortunately, the optical microscope comes closer to perfection than any other machine I know of. Resolution close to the maximum theoretically possible is obtainable, although expensive, with microscopes from several manufactures.
F. Also note again that the objective and condenser are the two most important components of the system. The oculars should be good enough that they do not degrade the image substantially while supplying further magnification, but normally do not have a major effect on the resolution of the microscope. II. Depth of field: A. Definition: The distance between the closest and furthest objects in focus at the same time. A lens system with a large depth of focus will be able to form images of overlapping structure simultaneously. B. Desirability (or undesirability) of a large depth of field: Where the specimen is arranged in a nearly planar manner, a large depth of field allows all objects to be in focus simultaneously. Fore example, this could be an advantage when differentiating cells is preferred. This allows fine optical sectioning and 3- dimensional reconstruction of complex objects such as cells (slide 1-52). In addition, the image at any given focus is clearer because objects immediately above or below the level of true focus do not impinge on the image. C. Factors that influence depth of field: 1. The numerical aperture of the objective: the higher the numerical aperture, the smaller the depth of field. Thus, good quality objectives tend to have a shallow depth of field. 2. The setting of the condenser aperture: the further the condenser diaphragm is closed the smaller the working condenser numerical aperture, and the greater the depth of field. (Note: this is another reason why the condenser diaphragm should be set up for Koehler illumination.) 3. The contrasting method used: DIC in particular, will tend to have a relatively shallow depth of field. III. Field of view: this one term has two separate meanings, normally you can pick out the proper meaning by the context. A. The trivial definition (see slides 1-55, 1-56): The absolute size of the specimen that can be imaged by a particular optical system. In this definition, a 20x objective will normally have a field of view twice as great as an 40x objective and half of that of a 10x objective AS LONG AS THE REST OF THE OPTICAL SYSTEM REMAINS THE SAME. B. The important definition (see slides 1-57, 1-58): The size of the image at the rear aperture of the ocular. This is related to the size of the virtual image that you see when you look through a microscope. A narrow field of view will result in the microscopist feeling that he is looking down a tunnel, while an exceptionally wide field of view will give the impression of being surrounded by the specimen. It turns out that is much easier to make and correct optics that will result in a smaller image at that plane, then one that will give a larger aberration-free image. This is one of the few recent major improvements in the light microscope. All parts of the microscope must be optimized to give a wide field including the condenser, the objective, the prisms, and most importantly, the ocular. Oculars that are designed to produce a wide field are frequently labeled wf. Older oculars so marked may have a 15 mm field of view; more modern systems can boast a field of view up to 30 mm. Putting a wide field ocular in an older microscope may not give a wider field if the rest of the optical system is not designed to transmit a wide field image to the ocular.
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Practical Hints
I. Changing magnifications A. Usually change by changing the objectives. However in rare cases you may choose to change magnifications by changing the oculars. B. The objectives are almost always placed in a rotating nosepiece. Always rotate the nosepiece by grasping the knurled surface -- NEVER BY GRASPING THE OBJECTIVES THEMSELVES. Rotating by means of the lenses could damage them. C. After changing objective lenses, the microscope must be readjusted for Kohler illumination. If the microscope is well aligned, you can simply adjust the field diaphragm until you see its image at the periphery of the field, and adjust the condenser diaphragm so that the light intensity is just short of maximum brightness. (Of course, by now you know that this works because the light will be at maximum brightness with the condenser NA greater than or equal to the objective NA. As the condenser NA falls below the objective NA the brightness will decrease. Optimal settings require that the condenser NA is slightly less than the objective NA.) For very critical work completely align as described previously. II. Determining magnifications: A. Definition: The magnification is the size of the image divided by the size of the object. Multiplying the magnifications of the ocular and objective lenses gives an approximate magnification. Here the image is a virtual image, not a real one, so the magnification is only approximate. The image is assumed to be located about 10 inches in front of the viewers eye. Obviously, this method is not satisfactory to determine the magnification of a photograph, and the magnification on a camera negative is likely to be much less than this. B. Use of a stage micrometer: To determine exact magnifications, you typically photograph a special slide (the stage micrometer) that has lines spaced at known intervals. Determining the negative (or video) magnification is a simple matter of dividing the line spacing of the image (measured) by the line spacing of the object (known). III. Cleaning the optics: A. The importance of clean optics (slide 1-60) B. By far the best way to clean optics is not to get them dirty. Every time you attempt to clean the optics you run the risk of scratching them. Additionally, some common cleaning agents can dissolve the cement that holds the glass lenses in the objective barrel. Probably as many lenses are ruined by cleaning attempts as any other way. C. If they must be cleaned, follow manufactures directions. Note that different manufacturers used different cements and coatings, so what works well in one case could ruin optics in others. If oculars etc. are dusty remove dust by (in order) blowing with an ear syringe, blowing with canned air, lightly brushing with a camels hair brush, or cleaning with a mild solvent (see below). The last should be done only as a last resort. D. Be especially sure never to get oil on one of the non-immersion lenses. (If you do so by accident clean immediately with solvent. E. Cleaning with solvent and cotton or solvent and lens paper: 1. Be sure you need to undertake this chore. 2. Be sure to use good quality cleaning materials. Lens paper is specially manufactured to contain minimal amounts of abrasive. Never use Kleenex which looks soft but in fact has abrasive particles inside. Likewise use good cotton and be sure it in unoiled (some drugstore cotton has oils added).
3. Before cleaning oil contaminated lenses, blot (NOT RUB) excess oil off the lens with clean lens paper. 4. Check with the manufacturer of the lenses and follow exactly what they recommend. Many of the solvents will dissolve lens cements and this will ruin lenses. Different cements have different solubilities in the various solvents so what is recommended may differ from one manufacturer to another. 5. Start with the most mild solvent possible and see if that will work before moving on to more aggressive solvents. In general, water is the least aggressive, ethanol or methanol next, with ether, xylene, benzene etc most aggressive. Some people use commercial lens cleaners and one even recommends Windex. Use only at a manufacturers suggestion. 6. The solvent should be applied to clean cotton or lens paper. Briefly brush over the element working from the inside out. Never rub as this will grind dust particles into the lens. Use each cleaning tool only one for a few seconds. This will minimize the damage that the dirt can cause the lens. Remember that a Q-tip or piece of lens paper is virtually free compared to the cost of a new lens. D. Cleaning interior surfaces (inner surfaces of lenses, prisms, etc.): 1. Dont. IV. Carrying the microscope: A. Use two hands. B. Do not invert the microscope, pieces may fall out. V. Storing the microscope: A. Keep out of dust. B. Do not store in humid area. Fungi can grow on the cement between the lens surfaces making the lenses cloudy. Such lenses are not normally repairable.
Contrast Methods I
I. Overview: A. The nature of the problem. Brightfield microscopy has as good a resolution as any other contrasting method, and fundamentally better than some others. However, resolution is only useful if objects can be seen, that is if they have enough contrast. An object that stops only 5% of the light rays that pass through it will seem invisible to the eye. Most biological objects have inherently low contrast. B. The solutions: 1. Stain biological objects (slide 2-1): We will say more about this later, but it has the obvious disadvantage that the most frequently used staining procedures require killing the specimens.
Using optical methods to increase contrast: These methods result in an amplification of one or more features of the light interaction with the specimen. We shall talk about the following: a) Darkfield (slide 2.2) b) Polarization c) Phase contrast (slide 2.3) d) Differential interference contrast 3. Using light emitted from the specimen itself. We will discuss this under Fluorescence Microscopy. II. The darkfield microscope A. Principle In brightfield microscopy, the image is made up predominately by light that has not been refracted or has been refracted very little. However, it is possible to exclude much of this light, and conversely collect a portion of the light that has been refracted. This is done by preventing those rays that normally pass through the specimen and directly into the objective from reaching the specimen at all. Instead light is focused on the specimen from such a direction that it will not enter the objective unless it is refracted. Thus, the only light that enters the objective has been refracted by the specimen (slides 2-4, 2-5). This results in a light image on a dark background - just the reverse of the brightfield image (slides 2-6, 2-7). B. Darkfield microscopy in practice: 1. To work, darkfield microscopy requires that the working numerical aperture of the condenser is larger than that of the objective. Objectives designed for darkfield may have a diaphragm built into the objective so that the working numerical aperture can be adjusted for best results (slide 2-5). 2. The direct (undiffracted) light is excluded from entering the objective by using a darkfield condenser (or a modified brightfield condenser) that illuminates the specimen only from the sides. The simplest way to do this is to put a darkfield stop immediately under the condenser (slide 2-4). This stop intercepts the light rays that otherwise would have directly entered the objective but its transparent edges allow light to illuminate the specimen from all sides. Special darkfield condensers reflect the central rays to the periphery for greater brightness (2-5). 3. Only a small percentage of light that enters the condenser will enter the objective, thus this method is very wasteful of light. Light intensity is often the limiting factor, especially at high magnifications (remember that the stop must block (or redirect) all the light that would have otherwise entered an objective of relatively high numerical aperture). 4. The theoretical resolution of darkfield microscopy is smaller than bright field microscopy because of the necessity to restrict the numerical aperture of the objective lens. C. How to identify darkfield images. 1. The surround should be dark 2. The specimen can scatter any color light. Therefore the specimen can be any color or combination of colors. 3. Gallery (slides 2-6,2-7)
2.
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III. The polarization microscope. A. Light and polarization: You saw this diagrammatic illustration of a light ray the first few minutes of the course (slide 2-10). I show it here to remind you that an electromagnetic wave is polarized, that is, that the wave has an up and down, right and left. Ordinary light has waves with all different orientations and the light is said to be unpolarized. However, it is possible to create beams of light in which all the waves vibrate in the same plane. Such light is said to be polarized. There are several ways to polarize light (slide 2-11 to 2-13). B. Polarization and interference. We have mentioned constructive and destructive interference. Now I can tell you that two rays can only interfere with one another when they are vibrating in the same plane (i.e. are polarized). You will remember that our drawings implicitly showed the two rays in this manner. You can get interference in unpolarized light only because the waves of each polarity interfere with the others of the same polarity. C. An aside: How do Polaroid sunglasses work? 1. Light reflecting off of water, a car hood etc. is polarized in the transverse direction. 2. The Polaroid in the sunglasses absorbs light polarized in that direction. Thus, the sunglasses absorb most of the reflected light. Unreflected light is unpolarized so the glasses will only absorb a portion (ideally 50%) of that light. The result is that most of the reflected light is absorbed, but only about half of the unreflected light. Of course, the glasses can also be pigmented to absorb more than the minimums mentioned above.) 3. Problem: What do you think you would see if you wear Polaroid sunglasses and were lying with your head parallel to the beach looking at the ocean? D. The effect of two polarizers: Definitions: If two polarizers are placed in series, the polarizer the light enters first retains the name polarizer while the second is now called an analyzer. 1. If the two polarizers have the same orientation, the analyzer has no effect because the polarizer has already absorbed the light vibrating in the appropriate plane. 2. If the two polarizers have an orientation of 90 degrees to one another, little if any, light passes through because the polarizer absorbs light of one polarity and the analyzer absorbs the light of the opposite polarity. Such an arrangement of polarizers is called crossed polars. 3. Any substance that rotates the plane of the polarized light can appear darker (if the polarizer and analyzer have the same orientation, and the substance rotates the plane of polarized light 90 degrees the substance would appear black on a gray background) or lighter (if the same substance was placed between crossed polars it appears gray on a black background). E. The polarization microscope: 1. Simply a cross between the situation we have just described and the bright field microscope. The microscope has polarizer between the light source and the specimen, and the analyzer between the specimen and eye. 2. Used to determine if objects rotate the plane of polarized light, or to study one or more of these substances. Objects that rotate the plane of polarized light are generally composed of many parallel subunits and are called birefringent (slides 2-14 to 2-19). F. Polarization in practice.
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1. Normally, the polarizer is between the field diaphragm and the condenser. The analyzer is usually in the body of the microscope (thus one filter will suffice for all objectives and all oculars). 2. Although the microscope can be used with many types of lenses, lens elements or the cement used to fasten them in place can rotate polarized light themselves, and can do so unevenly. Therefore, it is preferable to use special sf (strain-free) lenses that are created (or selected) for the ability to pass polarized light without affecting the direction or degree of polarization. 3. Specimens are usually viewed through partially crossed polars. Thus the specimen could be either darker or lighter than the background, depending on the direction it rotated the polarized light. 4. On microscopes specialized for polarization, the specimen is normally placed on a rotating stage. Therefore, the microscopist can place the specimen at any angle relative to the polarizer and analyzer. 5. In Biology, this technique is often used to study mitotic spindles and muscle fibers. However, it finds its greatest use in geology. C. How to identify polarization microscopy images. 1. The surround is often set to partially crossed polars. If this is the case, portions of the image may be darker than the surround, while others could be brighter. 2. The specimen can rotate different colors of light different amounts. Therefore the specimen often has multiple colors. 3. Polarization microscopy works with specimens composed of microscopic linear elements. Knowing if a particular specimen meets these criteria can give you a hint. 4. If a portion of the specimen is oriented differently with respect to the polarizer and analyzer, it will look different in the different orientations. This is particularly noticeable at right angles, where the specimen often appears light in one orientation and dark when it is at right angles to this orientation. 3. Gallery (slides 2-18 to 2-19)
Contrast methods II
I. General rationale for use of interference microscopy techniques (phase contrast, differential interference contrast etc. - see slide 2-21): When light goes from one substance to another with a higher refractive index, it slows down while traversing through that substance, only to speed up after leaving it. If the substance with a higher refractive index is clear, light rays will exit the substance with the same velocity, wavelength, and intensity as light rays that did not transverse the substance, but it will have an altered phase. The phase and DIC microscopes are designed to detect this difference. II. The phase contrast microscope (slide 2-22): A. General overview The phase contrast microscope blocks out most of the light that has not interacted with the specimen, while transmitting most of the light that has been slowed and refracted. After further slowing (usually) the refracted light, it is recombined with the undiffracted light leading to constructive or destructive interference between light rays of different types.
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B. Mechanism 1. A phase annulus (annular diaphragm) is present in the condenser and a matched phase plate is present in the objective (slide 2-23). The annulus illuminates the specimen with a hollow cone of light and the phase plate maximizes interference between the diffracted and non-diffracted light in the real (=intermediate image). The objective magnifies this real image exactly as it does in the brightfield microscope. 2. The mechanism by which the specimen, the annulus and the phase plate maximize interference is not difficult, but, for lack of time, we will not consider it here. Ask you instructor for details if interested. C. Characteristics of phase contrast: 1. The phase contrast microscope emphasizes small differences in refractive indexes between areas of the specimen. a) Therefore, it works very well for objects that have a refractive index only slightly different from the surrounding area (phase retarding capability of less than 0.1 wavelength). b) Because it amplifies differences in refractive indexes so dramatically, it does not work as well with specimens that have a very different refractive index from the surround. 2. Phase contrast creates rings around objects, which can make the image confusing (slide 2-27). These rings are particularly noticeable if the change of refractive index is relatively severe and becomes annoying if the object retards light more than about one half wavelength. It also makes measuring the size of objects inaccurate. 3. Phase contrast does not use the full numerical aperture of the optical system. Therefore resolution is actually worse than brightfield. 4. The image produced by the phase contrast microscope is artificial. It emphasizes changes in refractive indexes between objects. Usually this involves emphasizing edges of an object rather than the object itself. Further, the same component might look light if surrounded by one substance and dark if surrounded by one with a different refractive index. 5. The phase contrast mechanism reduces the image brightness about 98-99% compared to brightfield. Therefore it is important that the microscope light be much more powerful than is necessary for brightfield. 6. Phase contrast is considerably more expensive than brightfield, but not nearly as expensive as DIC. D. Phase contrast in practice. 1. It will only work with phase contrast objectives. (But you can use phase contrast objectives with other forms of microscopy with only a very slight compromise in optical quality.) 2. Usually have several different phase annuli in a turret under the condenser, so you can match the phase annulus to the objective (slide 2-28). 3. The phase annulus and phase plate must be aligned before use. a) Set up Koehler illumination b) Swing the proper phase annulus into position. c) Align the phase annulus and plate (slides 2-29, 2-30): (1) Visualize the phase plate and the phase annulus. Their images are at rear aperture of the objective. (This is the same plane at which you view the condenser diaphragm when you are setting up Kohler illumination.) To visualize them do any of the following:
(a) Insert the Bertrand lens and leave the ocular in place. (b) Or replace the ocular with a phase telescope . (c) Or remove the ocular and look down the barrel. (2) Move the condenser or the phase annulus centering controls so that the images of the annulus and the ring of the plate overlap. (3) Undo step 1 to get back to the image of the specimen. E. How to identify phase contrast microscopy images. 1. The surround is often featureless, but often less bright than brightfield 2. Borders are prominent. Areas where there is a marked difference in refractive index will often have rings. 3. Phase contrast microscopy works with specimens composed of very low contrast. Knowing if a particular specimen meets these criteria can give you a hint. 4. Gallery (slides 2-31 to 2-32; also see 2-46 to 2-48 to compare DIC and phase) III. Differential Interference Contrast (DIC) A. The name: Differential interference microscopy was invented by a German named Nomarski and his system was patented by Zeiss. Therefore, the term Nomarski interference contrast which is often used incorrectly to cover all types DIC, can only refer to systems made by Zeiss. Many other microscope manufacturers have similar systems that fall under the microscope manufacturers have similar systems that fall under the generic term DIC. They are sometimes named after the person who was responsible for working out the details for that company (i.e. Smith interference contrast). B. General principle (slide 2-35): Polarized light is split into two rays of polarized in opposite planes by a special modified prism. These two rays, travel through the specimen on separate but parallel paths. The two rays are very close together -- in the case of a 100 x objective the rays might be 0.22 m. apart. (Note the illustration in 2-35 shows them inches apart this is highly diagrammatic!) Because they are vibrating in different planes they can fit in nearly the same space without causing destructive interference. At boundaries one of these rays will travel through the region of higher refractive index and be slowed more than its companion. Thus, there will be a phase shift between the two rays. After passing through the objective, the rays enter a second prism and analyzer where they are recombined where constructive and/or destructive interference occur. The real image so produced is magnified by the ocular to form the final magnified image as in the other forms of light microscopy. C. Characteristics of DIC: 1. Objects appear in raised or sunken relief (slides 2-36, 2-37). 2. Traditionally image is presented with shadows as if by the setting sun. However, this is arbitrary, and shadows can be reversed by adjusting the prisms. 3. The apparent relief is not real, but an optical trick. What seems to be peaks are areas of different refractive index, and may in reality be perfectly flat or even valleys. Must use caution in image interpretation (slides 2-38 to 2-40). 4. Unlike phase contrast, halos are not present, this means DIC is superior for looking at edges of objects. 5. DIC uses the maximum numerical aperture of the condenser and objective. Therefore, the resolution of DIC can be as good as brightfield. 6. DIC allows very fine optical sectioning (see slide 2-41).
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7. Works best with objects of relatively large phase retarding ability (between 0.1 and 1.0 wavelengths). Thus is it complementary to phase which works best with objects of lesser phase retarding ability. 8. Works very well with video microscopy. 9. The DIC contrast mechanism reduces the image brightness by about 99% compared to brightfield. Therefore it is important that the microscope light be much more powerful than is necessary for brightfield. 10. Because DIC uses polarized light, it may not be suitable for specimens that rotate polarized light themselves. 11. The prisms and the strain-free optics are very expensive. This makes DIC much more expensive than the typical phase scope. D. DIC in practice: 1. The physical arrangement of the polarizer, analyzer and prisms vary tremendously from manufacturer to manufacturer. In most, a prism matched to each objective is installed in a rotating turret under the condenser. In most systems there is a single upper prism used for all objectives in the body of the microscope. The analyzer may be combined with the upper prism or separate. 2. Normally the positions of the polarizer, analyzer and first prism are fixed and the position of the second prism can be varied by the user to obtain the best image. E. How to identify DIC images. 1. Object appears in pseudo 3-dimentional relief. 2. The surround is often featureless, but often less bright than brightfield. 3. Borders are prominent, but often less so than phase contrast. 4. DIC microscopy works with specimens composed of low contrast and moderate phase retarding ability. Knowing if a particular specimen meets these criteria can give you a hint. 5. Gallery (slides 2-41 to 2-44; also see 2-46 to 2-48 to compare DIC and phase) IV. The Fluorescence Microscope A. Cf. fluorescence and types of microscopy we have talked about up to now: With brightfield, darkfield, polarizing or interference microscopy the specimen is imaged by differences in light passing through the specimen and light that passes through the surround. However, fluorescence uses light generated by the specimen. B. Principle of fluorescence. Many substances have the ability to absorb light energy and store it for a brief period of time. That energy has to go somewhere, and is normally released as heat, light, or a combination of the two. If the object releases light it is said to fluoresce. The photon of light released by the absorbing molecule cannot have more energy than the original absorbed photon, and usually has less, the remainder of the energy lost as heat. The energy of the photon is inversely proportional to its wavelength, therefore the fluorescence emitted by the excited molecule will invariably have a longer wavelength than the light originally absorbed. C. Fluorescence in biological objects: Many biological objects fluoresce naturally. Many others can be made to fluoresce by the addition of certain fluorescent tags (see slide 2-51).
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D. The transmitted fluorescence microscope: Not generally used anymore, but the principles of this microscope are easier to understand than the indirect fluorescence microscope (slides 2-52 to 2-54). 1. Light source: The light source for fluorescent microscopes generally produces a high intensity white light source. In this case it is directed upward towards the specimen and objective. 2. The exciter filter: This filter is designed to filter out all wavelengths except for those that will be absorbed by the object to be viewed (in this example blue). 3. The specimen: The object is illuminated with light of which it absorbs small amounts. After a short time the object fluoresces with the resultant light being of lower energy (in this case red) than the absorbed photon. The intensity of the fluoresced light is almost always orders of magnitude less than that of the exciting beam. 4. The objective: Both the fluoresced light and the stimulating beam not absorbed by the specimen enter the objective. 5. The barrier filter: The barrier filter is chosen to pass light of the wavelength of the fluorescence but absorb light of the wavelength of the exciting beam. 6. The ocular: As you should know by now, the ocular will further magnify the real image and produce the final image. E. The problem with the transmitted fluorescence microscope: Filters can be very good, but they are not perfect. The intensity of the signal is often very much smaller than the intensity of the stimulating beam. Thus even if the barrier filter can stop 99% of the photons of the stimulating beam, enough photons may get by to overwhelm the much smaller signal produced by the fluorescence (see slide 2-55). F. The epifluorescence microscope: Nowadays fluorescence microscopes are built on a slightly different plan (slide 2-56). 1. Basic construction (slides 2-57 to 2-58) The epifluorescence microscope, directs the exciting beam down onto the specimen through the objective. Thus, the majority of the stimulus beam that is not absorbed by the specimen passes the stimulus beam that is not absorbed by the specimen passes through the specimen and out the other side of the condenser. This means a brighter exciting beam can be used and that a dim signal can be observed without being overwhelmed by stray light of the excitation wavelength. a) The light source: The light source is usually a Xenon or Mercury arc because these sources have a wide range of wavelengths and are very bright. For less critical use, other sources are sometimes used. The light source is usually mounted quite high on the microscope. b) The exciter filter: This is exactly analogous to the exciter filter on the transmitted fluorescent microscope.
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c) The dichroic (dichromatic) mirror: This is what makes the epifluorescent microscope possible. A dichroic mirror passes light of some wavelengths and reflects light of other wavelengths. A particular mirror is chosen for exciter/barrier filter combination such that the mirror reflects light of the stimulating wavelength but passes light of the fluorescent wavelength. In our example, this mirror reflects blue light down through the objective to the specimen. d) The specimen: As we have seen the specimen absorbs a small percentage of the stimulating beam, the rest of the beam passing harmlessly through the slide. Some of the stimulating beam is reflected back through the objective and some of the fluoresced light also enters the objective. e) The dichroic mirror (again): The fluoresced light passes through the mirror, much of the stimulating light that has been reflected into the objective is reflected back towards the light source. f) The barrier filter: The barrier filter passes the fluoresced light, but absorbs the remaining light from the stimulus beam as well as light of other wavelengths that may have entered the objective. g) The image: The final image should consist almost entirely of light that has been fluoresced from the specimen. G. Characteristics of the epifluorescence microscope: 1. The most difficult part is sample preparation. 2. Lenses must be transparent to light of both the stimulating and fluoresced wavelengths. This is not always the case as the stimulating beam is often in the blue/U.V. range and many glasses and lens cements absorb in those wavelength. 3. Must choose proper exciter and barrier filters and the dichroic mirror (slides 2-60 to 263). Ideally, you pick a separate set for every fluorescent tag used so that the exciter filter passes most of the light of the stimulating wavelength (and little else), and the barrier filter passes as much fluoresced light as possible (and little else). The dichroic mirror should be chosen to reflect nearly all light of the stimulating wavelength but pass nearly all of the fluoresced light. Normally, the two filters and the mirror are sold in a set that can cost upwards of $1,000. In addition, the narrower the band of transmission, the more specific the signal, but at a reduction in brightness. This is particularly vexing because many samples have a very dim fluorescence. 4. For critical work, you must have a very bright light source. This is expensive, and must be aligned carefully (a non-trivial task). If you ever use one, be aware that the unfiltered light is dangerous to look at; it is very bright and filled with U.V. Most light sources should be left on for at least 30 min at a time, and never turned on while still warm from their last use. The bulbs are also expensive (about $150.00) and have a limited lifetime. 5. Because the objective acts as both condenser and objective, its numerical aperture is very important. A high numerical aperture objective lens will not only result in maximum resolution but also in maximum brightness. 6. There is no need of a condenser.
8. Is quite expensive. H. Epifluorescence microscopy in practice: 1. Particularly useful in indirect immunofluorescence (slides 2-61, 2-65). Here the investigator tags an antibody with a fluorescent component and allows the antibody to react with the antigen. By observing the pattern of fluorescence the investigator can determine the distribution of the original antigen in cells and tissues. 2. Operation of the fluorescence microscope is easy, but whoever is responsible for the microscope will be very protective of it. Be sure to follow his or her directions exactly as not to injure yourself or damage the microscope. E. How to identify fluorescence images. 1. Surround is normally black. 2. Fluorescent tags usually fluoresce over a narrow range of wavelengths. Therefore each tag will only be a single color. (Note that there can be multiple tags of different colors.) 3. Gallery (slides 2-62 to 2-65; also see 2-66 to compare fluorescence and phase)
Recording the image

I. Principle: Simple just replace eye with camera (slide 3-1). II. General considerations A. Consider sensitivity of the camera the more sensitive the better. Images are often not terribly bright, especially if contrasting methods such as phase or DIC are used. In many cases fluorescent images are very dim. B. The camera must be kept rigid on the microscope. 1. If the image is magnified 1,000 times then any vibration will also be magnified 1,000 times. 2. Thus if possible use a microscope with a photohead (often called a trinocular head), so that the camera can be on-axis of the optical train. C. If you are taking color images, you may have to worry about color temperature. Different light produce different colored white light. (See slide 3-2) Many light sources even will give different colors depending on how much current flows through them. (Thus turning up the intensity of the light might also cause the image to have a different background color.) III. Digital Microscopy Digital Still and Video Microscopy (see slide 3-3). A. Advantages: 1. Can use the maximum numerical aperture of both the objective and condenser lenses, which results in the maximum theoretical resolving power for the microscope. 2. Contrast can be raised electronically by the camera so the microscopist does not have to make optical sacrifices to maintain adequate contrast. 3. In most cases, the highest resolution digital images are monochrome. Software can be used to superimpose several monochrome images.
4. Digital microscopy allows interfacing with the computer. This allows: a) Subtraction of background image. b) Summation of many images for maximum information content. c) For motility studies, the investigator can electronically compare images at two times and subtract images of all objects moving. B. Disadvantages: 1. Good cameras can be very expensive ($30,000), although for many uses a much less expensive camera can be used. 2. For video, the framing rate is fixed because of video technology. It is very difficult to get more than 30 frames per second. For fast moving objects this is not enough (i.e. Chlamydomonas flagella make a complete beat in 1/60 sec.). 3. Printing out the image can sometimes be difficult.
Specimen Preparation
I. Rationale: Some biological specimens, e.g. unicellular protozoa, can be placed between a coverslip and slide and directly viewed by one of the forms of microscopy just mentioned. However, many interesting biological specimens cannot be treated in this manner (an elephant probably wouldnt stand for it). II. Common types of specimen preparation: A. Whole mounts (slide 3-4, 3-5): This is a common procedure if the specimen is small. In many cases the specimen must be first killed and preserved. Staining is optional, and the specimen can be mounted in a variety of different media. Examples of whole mounts include insects, fungi, protozoa, and parts of animals and plants. B. Squashes (slide 3-6): Frequently, it is desirable to look at cells of a tissue, without worrying about the relationship of cells to one another. The easiest way to do this is to squash the tissue between the coverslip and slide after which it may be stained and mounted. This technique is commonly used to study chromosomes. C. Smears (slide 3-6, 3-7): A smear is simply a means of spreading blood or similar bodily fluid over the slide, where it could be examined as is or stained and mounted. D. Sections (slides 3-8 to 3-10): This is probably the most general and useful technique for studying portions of complex organisms. The sample must be fixed, embedded, sectioned, stained and mounted. There is a bewildering array of specialized fixations, embeddings, and staining procedures; in fact entire courses are often taught covering only this subject. We will go over use of some of these steps as examples of specimen preparation. Remembers that considerations on fixation, dehydration and staining will apply to the other methods of specimen preparation as well.
1. Fixation (slide 3-11) a) Goals Fixation has two partially contradictory goals. On one hand, you want to avoid changing the shape, form or composition of the organism from what it was in life. This would argue for use of relatively gentle procedures. On the other hand, you must quickly stop all cellular activity and stabilize the specimen so it does not undergo degradation in the rather traumatic procedures which are to follow. Obviously the ideal can never be met, but methods are available that come reasonably close. b) Types of fixatives: (1) Heat: often suffices for bacteria etc. but does not preserve form well. (2) Coagulants: This type of fixative causes the proteins inside cells to congeal into fine strands. These strands may or may not be visible under the light microscope depending on the specimen and the quality of the microscope. For years, it was thought that the nucleus was filled with protein fibrils; we know now that these were artifacts caused by these fixative. Examples include the alcohols, chromic or acetic acid and mercuric chloride. (3) Non-coagulant fixatives: These substances cross link proteins and nucleic acids without causing coagulation. Examples include the aldehydes (formaldehyde, glutaraldehyde etc.) and osmium tetroxide. These fixative became popular after electron microscopy showed that they disrupt cellular structure much less than the coagulative fixatives. (4) Mixtures: Many traditional fixatives use a combination of several of the above substances, often containing both coagulant and noncoagulant fixatives. 2. Dehydration and embedding a) Dehydration: usually necessary because most common embedding materials are not water soluble. Most commonly, this is done by passing the specimen into gradually increasing concentrations of organic solvents. b) Embedding: This enables the specimen to be cut into sections. Traditionally, the sample is embedded into paraffin but today it is likely to be embedded into one of the plastics because a plastic embedded specimen can be cut into thinner sections. 3. Sectioning: An art. Typically the specimen is cut into sections 1-10 micrometers thick with a microtome and a glass or metal knife. 4. Staining: a) Also an art. Most stains are water soluble, so the paraffin must be dissolved by a hydration series. Generally, stains are colored and bind to specific components due to charge, size or differential solubility or an affinity for particular molecules (for example, IKI stains some starches) See slides 3-7, 39 and 3-100.) b) A special case: antibody-antigen. Although, this is really an affinity stain, it is so important it merits separate consideration. An antibody can have a very high affinity for its antigen and thus locate the antigen very specifically. The
label is usually an enzyme that makes a colored produce or a fluorescent molecule. E. A recent modification of the above fixation, embedding, sectioning and staining It is also possible to freeze a tissue and to section it in its frozen state. This is especially useful if the investigator wants to do enzyme or antibody localization on the tissue, because this procedure is less likely to destroy enzyme activity or antigenicity than the traditional fixation and embedding steps.
Introduction to Electron Microscopy

I. Disadvantages with electron microscopy A. Complicated (see slide 4-1): 1. Mechanically 2. Electrically 3. Operationally 4. Result: needs much more operator training, the microscope is considerably less reliable, it takes much longer to look at a specimen etc. B. Expensive: Both to buy ($100,000-300,000) and to maintain ($4-15,000 yearly, not including routine maintenance) C. Electrons do not penetrate well: Thus the specimen must be very thin. Almost all cells are too thick for electrons to penetrate. In addition, electrons will not even penetrate very far into air. This requires the specimen to be placed in a vacuum. D. Only non-living cells can be easily examined: Some experimental systems have been designed to examine living cells, but the best that can be done is to view dying cells or organisms. E. Therefore, one cannot show dynamic biological events. This is a very important limitation because most of the important biological questions involve changes over time. Such changes must be inferred from a series of static images (see slide 4-2). There is a certain amount of danger in this as sometimes there is no way to really check this inference. F. Specimen preparation is vastly more difficult: Due to the features a mentioned above, it usually takes days to weeks to prepare specimens for viewing. G. Very time intensive: Generally at least one person must do routine maintenance, cleaning, changing filaments, etc. In some places a person is hired full-time to take care of one or two microscopes. In addition, the microscopist using the microscope will generally have to spend considerable time to obtain good images.
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H. Limited field of view: This is a consequence of the higher magnification. At 100,000 magnification, the typical field of view will contain only about 0.01% of a typical cell cross-section. (And because the cell might be sliced into 200 sections, you are actually viewing less than one millionth of the cell at a time.) I. Electrons do not have color: Many of the pretty SEM pictures have been painted with one of those Hi-lighter pens or photoshopped (see slide 4-3). However, electrons do loose energy as they pass through the specimen and thus it is possible to generate pseudo-color in expensive analytical electron microscopes (slide 4-4, 4-5). II. The advantage of electron microscopy: A. Resolution (cf. slides 4-6, 4-7): Eye = 0.1 mm Light microscope = 0.2 um (a 500x improvement) Electron microscope = 0.1 nm (a 1,000,000 improvement on the eye and a 2,000x improvement on the light microscope. B. Theory behind the increased resolution: 1. Particles can be thought of as waves and waves as particles (slide 4-8). The wavelength of a particle can be stated wavelength = h(Planks constant)/mass*velocity 2. Substituting the known values for Planks constant and the mass of the electron and restating the velocity of the electron as a function of the accelerating voltage, the equation becomes: wavelength = 12/(accelerating voltage) 1/2 where the wavelength is in nm, and the accelerating voltage is in V. 3. An example: For a typical accelerating voltage of 100,000 volts, the wavelength of the electrons would be 0.04 nm or about 20,000 times smaller than light. 4. As we saw in the formula for the theoretical resolving power of the light microscope, the smaller the wavelength the greater the resolving power. The NA of the present day electron microscopes is not high enough to take full advantage of the lower wavelength of the electron, but the effective resolution is still about 2000x better.
The Electron Microscope

I. The electron gun: A. Purpose: The electron gun is designed to produce a bright focused electron beam with all of the electrons of the same energy (therefore the same wavelength). B. Design (slide 4-10, 4-11): A small amount of current (but large voltage) is passed through the filament. The electrons come off of the filament and are focused by the wehneld. At this point they have very low velocity, but are accelerated by the large potential difference between the wehneld and the anode.
C. Adjustment: The operator must be very careful in adjusting the current through the filament. At first, brightness increases with increasing current as more electrons are released from the filament. However, at some point (the saturation point), a further increase of current no longer results in increased brightness but does markedly shorten filament life. The microscope should be operated at, or just below, the saturation point. Changing the filament is expensive and takes hours of an instructors time. II. Electron lenses: A. Purpose: To focus the electron beam as glass lenses do for the light microscope. B. Design (slide 4-12): An electron lens uses a magnetic field to redirect electrons. They are basically composed of an electromagnet wrapped around a hole. Unlike glass lenses, the focal length (hence power), is changed by changing current. Thus, magnification and focusing are done electronically rather than mechanically as in the light microscope. All electron lenses are converging; thus diverging lenses cannot be used to correct for aberrations etc. as in glass lenses. C. Aberrations: Electron lenses, just like glass lenses have aberrations. 1. Chromatic aberration: The wavelength of the electron is determined by its energy. Therefore, electrons that acquire a different energy level due to loss of energy with the specimen or to an insufficiently regulated power supply will focus at a different plant than those of the main beam. This is a particular problem for thick specimens, because electrons will lose energy unevenly as they interact with the specimen. 2. Spherical aberration: This is a severe limitation on the resolution of the TEM because it cannot be corrected for by convex lenses, nor by making the lens aspherical. 3. Astigmatism: (slides 4-14 to 4-16) Astigmatism in the EM like astigmatism in regular optics means that the focal length of the lens is different in the different directions. It thus prevents a single focus point, and blurs the final image (slide 4-14a and b). Astigmatism is a constant worry, because astigmatism changes as the apertures (TEM equivalent of diaphragms) get dirty and as parts of the microscope age. Luckily, it is possible to electronically correct astigmatism. A stigmator is a ring of electromagnets which can be adjusted independently (slide 4-15). By varying the current to each magnet one can remove almost all stigmatism from an electron beam. The easiest way to do this is to focus on a hole and adjust the stigmators to give an even freshnel fringe (slide 4-15). 4. Other aberrations: Other aberrations such as curvature of field, distortion, and coma are not major problems in modern, well-designed and aligned microscopes. III. Image formation: The electron source and lenses function much like an upside-down light microscope (slide 4-18, 4-19). A. Electrons are produced by the electron gun and are accelerated into a beam by the anode. B. The electron beam is focused onto the specimen by means of the condenser lenses. This slide shows a single condenser lens; however modern microscopes almost always have
two lenses to allow for satisfactory brightness and resolution. Not shown in this diagram are the condenser apertures that are similar in function to the condenser diaphragm of the light microscope. C. The object is put into the electron path immediately above the objective lens. D. After interacting with the specimen, the electrons enter the objective lens. Unlike the light microscope, the electron microscope has only a single objective whose focal length is electronically changed to change magnifications. E. The intermediate lens has no real counterpart in the light microscope, but further magnifies the image produced by the objective. This lens is usually turned off when using the electron microscope at low magnifications. F. The projector lens is similar to the ocular in the light microscope. It magnifies the image produced by the objective and, if present, the intermediate lens. G. The final image. There are three ways in which the image can be formed: 1. The viewing screen. The human eye cannot see electrons directly, so the electrons are directed to a screen coated with cadmium and/or zinc sulfides. When an electron hits this screen, it emits a flash of yellow-green light that the operator can see through a glass panel. 2. Recording the image. Most modern microscopes have a hi-resolution digital camera. The image is recorded as a digital file and can be further processed on the microscope or using conventional software. Note: The image is your DATA. Any processing that one does beyond cropping, adjusting brightness and contrast must be spelled out and must not change the fundamental truth of the image. Anything else is academic fraud. H. Focusing (slides 4-21, 4-22): This is considerably more difficult in the TEM than the light microscope. The problem is that what looks good to the eye is usually not in the focus that will yield the most information. Underfocus gives the image with the most contrast and looks crisper than true focus, but results in less well-resolved objects. Most microscopists like to take pictures a little underfocused. The easiest way to do this is by finding a hole, adjusting it to find the underfocus ring, and go towards true focus until the ring almost disappears. I. A longitudinal section through a TEM (slide 4-23): This is what a cutaway view of the real microscope is like. Note the positions of the various lenses, specimen, screen and photographic plates. IV. The vacuum system: A. Purpose: Remember that electrons have very poor penetrating power, so an electron beam cannot travel far through air. Another reason for a high vacuum is more subtle; if an electron collides with an air molecule, the electron will scatter. Thus an electron beam will loose its focus and ability to for a good image. The vacuum system thus removes air from the microscope column at several points (slide 4-23).
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B. The mechanical pump (fore pump) (slide 20): The mechanical pump can rapidly move a lot of air out of the microscope column by the method shown. However, such pumps cannot give the high vacuum necessary for satisfactory performance. C. The diffusion pump (slides 22-25): This is the most common mechanism for obtaining the ultimate vacuum. In this pump, hot oil is boiled and moves up into the chimney of the pump. There it is cooled, condenses and moves outward and down. The key to the pumps effectiveness is that this downward stream is very rapid and carries air molecules down with it. The air molecules are thus concentrated at the base of the pump and removed by the mechanical pump. This type of pump works very well, but it is very sensitive to mistreatment. If the cooling water stops, hot oil is spewed into the interior of the super clean microscope, a process called backstreaming. Backstreaming can also occur if the mechanical pump shuts down or if the valve between the mechanical and diffusion pumps is closed. Such a catastrophe requires weeks of work to clean up. Dont ever let it happen to you. D. The tubomolecular pump (slide 4-32). This is basically a series of very high speed fans that whaps the molecules of air from the column. It is very fast, although it does not remove water vapor as well as most other molecules. Because the blades rotate so rapidly, the bearings must be very good (bearing failures have been known to occur with messy results). This is the kind of pump we have on the SEM. This pump is considered clean (no oil contamination), but still has to be treated carefully. It is more expensive than the diff pump and needs maintenance.
EM Specimen Preparation
I. Whole mounts: A. Specimen support: A specimen thin enough to be electron lucent, is normally small enough to fall through the holes in the copper grid. Therefore, a very thin, electron transparent film is normally applied first. Usually this film is composed of a plastic film (formvar or colloidon) which has been cast onto a glass slide and floated off onto water. For critical work, a layer of C is placed over the plastic. C is electron transparent and has the added advantage of being conductive so it will conduct electrons away from the specimen and prevent charging. For more critical work, the plastic can be dissolved away after C coating. For the ultimate in resolution, you can make a holey C film and look through the holes. B. Types of specimens: The specimens must be thin, and lack water. Generally these specimens consist of parts of cells (eg chromosomes or diatom frustules, see slides 4-36, 4-37). Some cells have exceptionally thin edges that can be imaged in the scope, but very few cells can be examined in their entirety (slide 4-38). II. Positive stain: A. Principle (slide 4-40) Here the specimen absorbs some substance that blocks electrons. B. Uses: The positive stain is rarely intentionally used but some images of positively stained molecules and subcellular particles can be found in the literature.
III. Negative stain: A. Principle (slide 4-40): Here the object is surrounded by stain. The object itself is clear on a dark background. This is an easy technique and it will give high quality images. As a consequence it is very common. B. Procedure: Simply place specimen on grid, cover with an aqueous solution of a heavy metal salt, remove most of the stain solution, and allow the rest to dry around the specimen. C. Uses: Negative stain is extensively for microorganisms (slide 4-41), viruses (slide 4-42), subcellular particles, and molecules (slide 4-43). IV. Shadowing: A. Principle (slide 4-45): An electron dense layer is evaporated onto the specimen, usually from one side. The metal layer piles up against objects and is absent from their shadow. Under the microscope, the heavy metal absorbs electrons, the sample being effectively electron transparent. This procedure normally results in dark objects and white shadows, but some people reverse the contrast to give an image that is more normal looking. B. Procedure (slides 4-46 to 4-48): The specimen is placed in a vacuum evaporator, and electrodes are set up in such a way as to allow a current to heat the heavy metal. After the specimen and metal source are placed in the correct geometrical arrangement, the vacuum chamber is evacuated and the current applied. After the heavy metal has been evaporated, air is readmitted into the chamber and the finished grids are removed. C. Uses: Used for microorganisms (slide 4-49), subcellular particles and molecules (slide 4-50). It is extensively used to image nucleic acids (slide 4-51). D. A modification: low angle rotary shadowing. Here the specimen is rotated as the metal is evaporated. This doesnt result in a shadow, but the metal does pile up at the specimen. The metal atoms tend to move after deposition to form granular aggregates. This is used to show the structure of nucleic acid and protein molecules (slides 4-53, 4-54). V. Freeze fracture: A. Principles (slide 4-55, 4-56): This is really a specialized form of shadowing, but it is important enough to be considered by itself. Here the specimen is frozen, and then placed in a freeze-fracture, freeze-etch machine. This machine is similar to the vacuum evaporator with the added feature of being above to keep the specimen at about -100 to -150 degrees C. The sample is then cracked with a cold knife while under vacuum. The cracks tend to go between the leaflets of any membranes close to the path of the crack. The specimen is immediately shadowed with a heavy metal. It is this step that will provide the contrast under the TEM. A relatively thick carbon layer is applied next; this carbon layer is necessary to hold the resultant replica together through subsequent processing. Carbon is chosen for this role because it is nearly electron transparent. After creation of the replica, the sample is removed from the machine, and the specimen itself is dissolved. Lastly, the cleaned replica is picked up on a grid and placed into the microscope to be viewed.
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B. Procedure: The exact procedure is complex and very dependent on the specimen and the type (an age) of freeze-fracture machine. If you were to contemplate using this technique, get training from someone who uses it regularly. C. Uses: The freeze-fracture technique is used extensively to study membranes structure. membrane fusion, etc. (slides 4-56, 4-57), because the cleavage plane normally passes through the centers of the membrane. It is rarely used for studies that do not involve membranes. Freeze fracture and thin-section are compared in slide 4-58. D. Caution: This procedure gives a three-dimensional image. However, whether the image projects towards or away from the observer is largely determined by the brain processing the image, rather than by real information present in that image. Simply turning the image over is likely to give the impression of the opposite orientation (slide 4-59). Therefore, as a microscopist you must be very careful in presenting the image to give the right topographic impression, and, as a viewer, you must be skeptical in accepting such images as a representation of reality. VI. Freeze-fracture, freeze-etch: A. Principle (slide 4-60): The procedure is in all respects similar to freeze fracture, except that after fracture the specimen is warmed to about -100 C for a few min. Under a good vacuum, some of the ice will sublime (etch) away from the specimen, allowing biological objects to project out of the ice. After this the replica is made in the normal way. Where freeze-fracture gives images of the inside of hydrophobic objects such as membranes, freeze-fracture, freeze-etch gives images of any non-etchable structures. B. Procedure: Even more difficult than freeze-fracture. Get help before trying. C. Uses: Gives three-dimensional images of subcellular organelles, particles cytoskeletal proteins, etc. in situ (slides 4-61, 4-62). VII. The Thin-Section Technique. A. Introduction: Thin-sectioning is by far the most widely used specimen preparation procedure in transmission electron microscopy. We will, therefore, spend more time on this subject than the others. I would remind you that some sections of this section also are relevant to other techniques previously discussed; for example, the specimens might be fixed before freeze-fracture with fixatives commonly used for thin-section. B. Overview (slide 4-65): C. Fixation: 1. Purpose: Like fixation for the light microscope, the fixation procedure is designed to preserve structure as close as possible to the structure present in the living organism. It is also important the fixation procedure stabilized the specimen through subsequent steps in processing. 2. Contents of fixative solutions: Solutions used for fixation contain a fixative agent which cross-links macromolecules, a buffer to stabilize the pH, and one or more substances to ensure the proper osmotic balance.
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3. General comment on fixation protocols: The optimal fixation procedures are strongly dependent on the organism and the goal of the investigator. There is an unbelievable amount of black magic in choosing the proper pH, time, temperature, fixative agent(s), type of buffer, osmotic strength, etc. As an investigator, the first thing to do when choosing a protocol is to look in the literature for good images of samples similar to those you wish to prepare, and follow the method that seemed to work best. This is not guaranteed to give you good results, but it is a starting point. Also, it is important to use as small a sample as possible to ensure adequate penetration of the fixative. 4. Common fixatives: a) Glutaraldehyde (HCO-CH2-CH2-CH2-HCO): (1) Advantages: (a) Results in good preservation, especially of microtubules, vacuoles, vesicles, ribosomes, etc. (b) Is a rapid penetrator. (c) Does not always denature enzymes and epitopes. (2) Disadvantages: (a) Does not stabilize lipids or carbohydrates. (b) Denatures some epitopes and enzymes. (3) Comments: Glutaraldehyde is the universal first fixative. It makes very strong (essentially irreversible) cross-links between protein components. Pay close attention to pH as glutaraldehyde will polymerize above pH 8. b) Formaldehyde (HCO-CH3): (1) Advantages: (a) Faster penetrater than glutaraldehyde. (b) Less likely to denature sensitive epitopes and enzymes. (2) Disadvantages: (a) Does not cross-link as well as glutaraldehyde. (b) Cross-links are reversible. (3) Comments: Formaldehyde is extensively used where speed of penetration is important, often in a mixture with glutaraldehyde. In addition it is used in immunolocalization or enzyme localization where the activity of a nondenatured protein is important. c) Osmium tetroxide (OsO4): (1) Advantages: (a) Fixes lipids and fatty acids. (b) Imparts some contrast to the tissue. (2) Disadvantages: (a) Dangerous. (b) Will not fix carbohydrates, nucleic acids (c) Will not by itself fix many important cellular structures such as microtubules etc. and can even break down previously stabilized actin filaments. (d) Denatures everything. (e) Is a relatively slow penetrator. (f) Expensive.
(3) Comments: Most of the disadvantages of osmium are mitigated by its normal use as the universal second fixative. It is also used as the first fixative in a widely used fixation method for bacteria. d) Uranyl acetate: Sometimes used as a fixative, it is usually more important as an en bloc stain (one that is applied before sectioning). e) Others: There are a number of other fixatives such as acrolein (tear gas). Most of these are special purpose and will not be mentioned here. D. Dehydration: Most embedding media are hydrophobic, so the specimen must be transferred into an organic fluid such as alcohol or acetone. Normally this is done via a graded series to minimize damage done to the specimen. E. Embedding and polymerizing: Most embedding is done in unpolymerized epoxy resins (plastics). The sample is transferred into a graded series of the unpolymerized resins over a period of hours to weeks. After the transitional fluid has been completely replaced by unpolymerized epoxy, the epoxy is heat-polymerized over hours to days. The final result is a plastic block with an embedded sample ready to be taken to the microtome (slide 53). F. Sectioning: This is another art in electron microscopy. The specimen must be sliced into sections 50100 nm thick. To give you an idea of how thin this is imagine slicing a piece of paper from 2,000-4,000 times thinner! 1. Block trimming: A small sample is more easily sectioned than a larger one. A good size is about 0.5 mm square (slide 4-66). 2. Ultramicrotomes (slides 4-67, 4-68): These machines have an incredibly precise advance base either on controlled heating of a metal rod or on a system of cams and levers. The block is mounted in the chuck of the microtome. 3. Knives: Obviously a sharp knife is absolutely essential for obtaining adequate sections. Metal knives cannot be sharpened adequately so other materials are necessary. a) Glass knives (slide 4-69): (1) Advantages: (a) Can give good results. (b) Are relatively inexpensive. (2) Disadvantages: (a) Difficult to produce good knives every time. (b) Even the best knife is not good along the whole edge. (c) Glass flows, therefore knives must be made up immediately before use. (d) Must make boats to allow for flotation of specimens. These boats are notorious for leaking just before you are ready to collect the sections. (e) Very fragile.
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Sectioning itself destroys the knife edge. Therefore, even under the best conditions, you can only get a few good sections before you have to move the knife and start over (slide 4-70). b) Diamond knives (slide 4-71): (1) Advantages: (a) Can cut consistently good sections. (b) Are only very slowly dulled by proper cutting of the block. Therefore you can cut thousands of sections from the same part of the knife. (c) Have built in boats that never leak. (2) Disadvantages: (a) Expensive: prices are around $1,000 per mm of cutting edge. (b) Fragile: simply touching the edge will ruin it. c) Sapphire: (1) Advantages (a) Cheaper than diamond (b) Better than glass (2) Disadvantages: (a) Will not last nearly as long as diamond. (b) Much more expensive than glass. 4. The process of sectioning: The boat is filled with water, and the block is brought to the knife. The block is slowly advanced to the knife until, sections are cut off the block to float upon the water (slide 4-72). The section thickness can be estimated by looking at the interference colors of the sections. When good sections are finally obtained, they are picked up on the sample grid. G. Staining: 1. Purpose: The sections as cut probably have little or no contrast. The sections are then stained to bring up the total contrast of the specimen. Additionally, some stains will stain different components of the specimen differently, giving the microscopist some idea of the chemical composition of parts of the specimen. 2. Common stains: a) Uranyl acetate: This is usually the first stain employed. It will stain the nucleic acids well, proteins less well and lipids and carbohydrates not at all. It gives only a moderate level of contrast. b) Lead citrate: This is usually used after uranyl acetate. It is especially useful for specimens that have been fixed with osmium. It stains RNA containing nucleoproteins and most other proteins with a high contrast. It is sensitive to atmospheric CO2 and give rise to lead dirt which is always bothersome and occasionally can obscure the specimen. 3. Staining procedure: The grids are simply floated on drops of the relevant stain for a few min. and rinsed well in distilled water.
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H. Use: Thin-sectioning is used with almost all types of biological specimens larger than molecules (slides 74, 4-75). I. Useful modifications of the thin-section procedure: As in light microscopy, a wide range of specialized staining procedures are available. One of the most useful is stains based on immunocytochemical methods. For example, small gold beads can be bound to an antibody molecule and the resultant reagent will specifically seek out its antigen. This is a very powerful tool but fraught with experimental difficulties. Potential problems include: denaturation of the antigen due to fixation, denaturation of the antigen due to dehydration and/or embedding, accessibility of the antigen embedded in plastic, non-specific binding, and other. Never-the-less it is such a powerful tool that it is being used more and more extensively (slide 4-76). J. Interpretation: Thin sectioning poses two particularly vexing problems. 1. Sections are too thick: Even though the sections are only about 50 nm thick, this is relatively large compared to some cellular structures (i.e. 2-3 x as thick as microtubules or ribosomes, 5 x as thick as actin filaments and 25 x as thick as the DNA molecule. Therefore, an EM image is largely composed of overlapping structures which must be sorted out. 2. Sections are too thin: It is very difficult to really understand the three dimensional organization of cells or organelles in a section that is of random orientation and very thin compared to the total object.` VIII. Ultra-rapid freezing: A. Principle: Ultra-rapid freezing is perhaps the most significant advance in biological specimen preparation of the last 15 years. Both normal freezing and conventional thin section techniques can change the specimen in unknown and unpredictable ways. Basically, the problem with the traditional freeze-fracture, freeze-etch occurs at the very first step. Conventional freezing methods freeze relatively slowly and can allow ice crystal growth. This is sometimes minimized by pretreatment of the sample, but this pretreatment is likely to change the specimen. Likewise the chemical fixatives used in conventional thin section microscopy have been shown to change the structure of the specimen itself. The ultra-rapid freezing techniques tries to get around these limitations by freezing at a rate of at least 100,000 degrees C/sec. After rapid-freezing, fracture and etching can be carried out normally. B. Methods of rapid freezing: 1. Slamming (slide 4-77): In this technique, the object is slammed into a metal mirror kept at liquid nitrogen, or preferably, liquid helium temperatures. In a good slam, the specimen may be well frozen to a depth of 10-20 um (about 1 cell diameter). The fracture must be made through this area. 2. Plunging: In this technique, the sample is usually spread into a very thin layer and plunged into a liquid held at as low a temperature as possible. The thin layer makes fracture difficult, but some specimens can alternatively be placed in a cooled stage in the electron microscope and viewed directly.
3. High-Pressure: High pressure inhibits ice crystal formation, so in theory a sample could be better frozen under pressure. All kinds of methods have been tried (including pressurizing the sample with a rifle slug), and there is one devise presently on the market. This technique shows promise, but it is expensive, difficult and has not been shown to be free of artifacts. C. Processing of the sample after freezing: 1. The sample can be treated by the normal freeze-fracture, freeze etch procedure. This gives a three dimensional relief image of the sample. The technique can also be adapted to study individual molecules. 2. Alternatively, the sample can be freeze substituted. Basically, this involves fixing the sample in a frozen state and then dehydrating and embedding it for conventional thin-sectioning. This is very useful for organisms or subcellular components that do not preserve well with the conventional methods. D. Uses of rapid freezing: The technique has become very important in the study of fast biological phenomena such as neurotransmitter release, actin-myosin interactions etc. It is also extensively used to study cytoskeletal or structural proteins (slide 4-78). The technique can also be adapted to study individual molecules (slide 4-79). It is an invaluable method to check-up on results obtained by more conventional means. If the two methods give vastly different results it is an indication that something is wrong with one of the methods.
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The Scanning Electron Microscope

I. Principle behind microscope (slides 4-83, 4-84): The scanning electron microscope is similar to the TEM in many respects. Like the TEM, the SEM produces and focuses the electrons and the image is comprised by the interaction of the electrons with the specimen. II. The SEM: A. The electron gun: Almost exactly like the gun from the TEM, except that it is typically operated at a lower accelerating voltage. B. The condenser lenses: Like those of the TEM, the condenser lenses focus the electron beam. A good set of condenser lenses will focus the beam to a spot about 100 um (0.1 mm). C. Scanning coils: These coils are capable of moving the entire electron beam in a grid shaped pattern. Thus it is the scanning coils that are responsible for the way the image is created in the SEM. D. Probe forming lens (=objective lens): This lens is very different from the objective lens in the TEM. For one thing, the electron beam goes through this lens before hitting the specimen. In addition the probe-forming lens makes the electron beam smaller in the SEM, but enlarges the image in the TEM. In a good SEM the objective can focus the electron beam down to about 3 nm. E. The electron beam scans across the specimen in a regular array with exquisite accuracy. At any one time, the beam is hitting only a small portion of the specimen. Secondary electrons are emitted from the specimen at that point (and only that point) with a very low velocity. F. The detector (slide 4-85, 4-86): The detector is designed to measure the number of secondary electrons emitted at each position of the electron beam. It detects them in the following manner: 1. The electrons are attracted to the positively charged grid that overlies the detector itself. 2. These secondary electrons then bombard the scintillator, a specialized structure that converts the electrical signal to a light signal. 3. The light signal is transmitted out of the microscope column by the light pipe. 4. At the other end of the light pipe, the light is changed back to electrical energy by the photocathode. 5. This electrical signal is amplified and in concert with the electrical signal produced by the scanning coils, sent to an output. G. Image formation: The detector measures the number of secondary emitted electrons for each position of the beam (and hence part of the specimen), and displays this information on the monitor. The more secondary electrons that are emitted by a particular portion of the specimen, the brighter the image is at that point. Therefore, the final image is really a map of the relative secondary electron emissions. Luckily, the emission and subsequent detection of secondary electrons is determined by the shape and position of the specimen, so that this map is very similar to the outside shape of the specimen.
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III. Specimen preparation: A. The requirement for specimen preparation: A few biological specimens can survive the vacuum and electron bombardment for a short time. Thus, it is possible to view insects moving around. However, most specimens must be carefully prepared before they can be examined and preparation will improve the images of all biological specimens. B. Fixation and dehydration: These steps are very similar to those of the TEM, except the times of each step can often be shortened for SEM preparation. In addition, larger samples can be fixed for SEM, because only the surface of the sample will be examined. C. Drying: This is the most important step in the procedure, and the one most likely to lead to problems. Listed below are the common forms of drying: 1. Air drying: This procedure is easy, quick and cheap. Unfortunately, surface tension is very strong over the short distances that will be observed with the SEM and thus is likely to destroy all but the most robust portion of the specimen (slide 4-87). This technique can be acceptable for specimens that have low water content to start with such as wood samples or insect exoskeletons. 2. Freeze-drying: This procedure can give good results, especially in specialized cases. Surface tension effects are minimized because the ice can be sublimed away without ever becoming a liquid. 3. Critical point drying: This is the generally preferred way to dry biological samples. a) Principle: The critical point is the temperature and pressure above which there is no difference between liquid and vapor (slide 4-88). Therefore, at or above the critical point, a liquid can turn into a gas and exit the sample without causing surface tension effects. b) Practice: The sample is transferred into the critical point apparatus (slide 4-89) and infiltrated with a solvent that has a reasonable transition point, commonly CO2 (transition point = 73 atm (1060 p.s.i.) at 31.1 C). The temperature and pressure inside the apparatus is then raised above the critical point, and the gas allowed to slowly bleed off. Finally the specimen is removed from the apparatus and mounted. D. Mounting: The sample is then mounted onto a suitable support called the stub (slide 4-90). E. Coating: The specimen is normally coated with a layer of conductive material to prevent charging under the electron beam. If the layer is also dense, it will ensure that the secondary electrons are emitted from the surface of the specimen and will also screen the detector from electrons that are backscattered from deeper in the specimen. Thus the image obtained will better reflect the surface geometry. The most common conductive layer is a thin layer of gold that is produced in the sputter coater (slide 4-91).
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IV. Uses: In biology the SEM is most frequently used to create nifty picture of bugs or to discover more surface features of organisms as an aid to their taxonomy. It is also used in all fields to examine surface detail (slides 4-92 to 4-94). V. A recent modification: Very recently, there has been an important development in the SEM. Some SEMs now have the capability of imaging partially hydrated specimens in a low vacuum state. This makes specimen preparation much less difficult and allows examination of living (actually dying) specimens (slide 4-96, 4-97). In addition, the water present in the sample can conduct electrons away from the specimen and reduce charging. This partially alleviates the requirement for coating with a conductive layer such as gold. This trick is managed by designing the SEM so that several areas with different partial pressures can be maintained simultaneously. The electron gun is operated at high vacuum (about 10-90 ATM) while the specimen chamber is at about 5% of ATM. These are presently specialized and expensive instruments, but in time they could become more widely used. Slide 4-98 gives an example of this kind of image.
Other Forms of Microscopy

I. Recent advances in light microscopy (Slide 5-1) A. The Confocal Light Microscope (slide 5-2, 5-3): The original microscope was invented by a Czechoslovakian scientist about 50 years ago, but there was no money to develop the microscope and no one in the west knew about it. After its "discovery" by western scientists, it has become a "hot" instrument and many companies are making updated versions. This type of microscope has the ability to take very fine optical sections including specimens that are otherwise difficult because of flair or because they are only partially transparent. In its simplest form, it works by passing the illuminating light through a hole in a spinning disk and thus illuminating an arc of the specimen. The image is built up in a series of arcs much like an SEM image. However, the real secret is that the light must pass through a second hole in the disk before forming the image. Any light that is scattered by the specimen or that illuminates the specimen in a plane other than the focal plane misses the hole and is eliminated from the image (slides 5-4 to 5-7). Many newer instruments use a laser as the light source and a single hole that allows light from the proper 3-dimensional point to reach the detector while excluding out of focus light. The confocal microscope can result in a very slightly improved theoretical resolution over the traditional light microscope, but its real advantage is that light from out of focus components does not interfere with the final image. This is particularly useful in the viewing specimens embedded in inconvenient matrixes such as nerve cells in a tissue, fossil insects in amber (slide 5-8), or the inner layer of an intact skull. Another area where it is exceedingly useful is in fluorescence microscopy of three-dimensional objects (slide 5-9 to 5-11). Remember that the traditional the stimulating beam of the
traditional fluorescence microscope bombards all areas of the specimen at once. Portions of the specimen that are out-of-focus will produce a fluorescence signal just as readily as portions of the specimen that are in-focus. The resulting image will therefore be composed of both in-focus and out-of-focus components. However, in the confocal microscope the images is built up by a collection of points where only light emitted from the in-focus point will be collected. The image built up from a collection of such points will therefore be a true optical section through the object. Because, the microscope is essentially getting the intensity of light from a collection of x-y-z coordinates, images are typically generated in a computer. This allows exceptional 3-dimentional images to be built up. These microscopes are very popular among those few people who can afford them and can be considerably complex to operate. A. Super-resolution microscopy: 1. A reminder of the physics of resolution (slide 5-13): Remember that there is a limit to the resolution that can be obtained with light that depends on the wavelength of the light and the numerical aperture of the system, This is enforced by interference effects of the light as it passes through closely spaced portions of the specimen. 2. A single infinitely bright and infinitely small object will generate an image called an Airy disk (or Airy disc; this term is based on the discovers name rather than being descriptive). The Airy disk is sometimes referred to as a circle of confusion, because the conjugate point of the image is inferred to lie within it, but one cannon see the image of the actual object (slide 5-14). 3. No matter how small the actual object is, its image cannon be smaller than the Airy disk (slide 5-15). In fact it is the size of the Airy disks that limits the resolution of the light microscope. 4. Setting up the microscope well (slide 5-17) allows the smallest possible Airy disks (low wavelength and high numerical aperture), but diffraction effects limit the possible resolution for normal images formed by lens systems. 5. In complex specimens detail is lost because of overlapping circles of confusion (slide 5-18). 6. If one can image only one (infinitely small, but very bright) object at a time, one could infer it probable position within the circle of confusion. Better sampling (more light measured over longer time periods) allows greater statistical certainty about its actual position (slide 5-19). 7. So one type of superresolution microscopy involves doing the above for every spot on an image. This works with a set of fluorescent tags that are very small and very bright. In addition, the tags can be turned on and off. So the general strategy is to turn on a very few tags (so that you are unlikely to have overlapping circles of confusion), and determine where each point source is likely to be within their respective circles of confusion (slide 50-20). This gives you very accurate positions for a few tags at a time. The one turns off the first set of tags, and turns on another small random set. Photons are in turn collected from this second set, and the likely positions of these tags are determined as above. Then the process is repeated until an image is built up (5-22 and 5-23). 8. The disadvantages of this method are: a) It takes a very long time to create an image. In many cases it take all day for one image. This obviously means one must used fixed (non-living) samples and cannot view dynamic events.
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There are a limited number of tags that are small, bright, stable (that is do not photobleach), and that can be turned on and off at will. c) It is very expensive. 9) A competitive form of superresolution is called Synthetic aperture. This is a form of scanning light microscopy in which advanced optical techniques are used to illuminate only a tiny portion of the specimen at a time, and to collect photons from only that portion. We will not talk about that further here. II. Analytical electron microscopy A. Overview: So far we have only considered a very few of the possible outcomes of the interaction of the electron and the specimen. In the TEM we have used only the difference in the intensity of the electron beam (percentage of electrons which penetrate without being deflected) as it passes through various regions of the specimen. In the SEM we have considered a different class of electrons, the secondary electrons that are knocked free from the specimen as a result of bombardment with the electron beam. However, there are many other possible interactions of the specimen with the electron beam (slide 6-1). These include the elastically scattered electrons, the inelastically scattered electrons, backscattered electrons, secondary electrons, auger electrons (pronounced O.J.), and the production of X-rays or light. Each of these has potential information about the structure and composition of the specimen. B. An example of an analytical electron microscope: the STEM. Some of the following analytical methods can be accomplished on the traditional TEM, the SEM or both. However, other analytical techniques can be done only on the scanning transmission electron microscope (STEM). The STEM is designed much like a TEM where the principle mode of observation is the transmitted electrons. However, the electron beam is scanned over the specimen in a grid-like pattern, much like that of the SEM. Thus, in the STEM mode, only a very small portion of the specimen is irradiated with electrons at any one time. This means that all electron diffraction, x-ray or light emission, secondary electron emission, or scattered electrons produced at any point in time must have come from that point of the specimen. This microscope can therefore be fitted with a truly exceptional number of different detectors (slide 6-2). The STEM can make an image with secondary electrons similar to an SEM, or transmitted electrons similar to a TEM (slide 6-3), but can also be used for many other different analytic methods (below) C. Analytic methods: 1. STEM analysis of scattered electrons (slide 6-4): In the standard TEM, the image is composed of non-scattered electrons (making up the bright background) minus the electrons that are absorbed or are scattered by the specimen (thus giving rise to the dark object). However in the STEM it is possible to collect only the scattered electrons. This will give a darkfield image. In addition, it is possible to measure the number of electrons scattered by any portion of the specimen. Because the scattering of the electron beam is proportional to the mass with which it is interacting, it is possible to determine the masses of large biological molecules and their subunits individually (a direct measurement of their molecular weight). There is an instrument of this type at Brookhaven National Laboratory. If you have such a problem you can probably get a grant to use it. 2. Electron diffraction (slide 6-5):
b)
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3.
4.
5.
B.
Suppose that the specimen is composed of very small subunits. Some of the electrons that interact with the each subunit will elastically scattered, that is, their trajectories will be changed (with no loss of energy). The direction of the change will be determined by the characteristics of each subunit. If the subunits are arranged with a repeating pattern, then each subunit will scatter electrons along a similar path, and these electrons will make up a pattern that can be photographed. The positions of the electron spots are determined by the properties of the subunits. These electron diffraction spots can give direct information about the size of the subunits and their degree of order. In addition, the diffraction image can be mathematically converted into a two or three-dimensional reconstructed image of the subunit. This may sound like a roundabout way of determining structure, but is usually much more accurate than can be obtained by direct visual observation (assuming proper technique and a well ordered specimen). However, this technique requires crystallized or otherwise very well ordered specimens, a great deal of expertise, a good set of computers and a lot of time. This technique can be carried out on most TEM's and STEM's and some SEM's. X-ray analysis: When an electron hits an atom in the specimen it sometimes knocks out one of the inner electrons. When this happens, an outer electron can fall into the hole left by the departure of the inner electron releasing excess energy in the form of an X-ray (slide 6-6). Each element will release X-rays with characteristic energies (= characteristic wavelengths). Thus with the proper X-ray detector it is possible to determine the atomic composition of the sample (slide 6-7). This technique works best with the heavy elements (atomic weight greater or equal to that of Si), because the x-rays from these elements are energetic enough to penetrate the detector window, but can in exceptional cases identify much lighter elements. This technique can be carried out with suitably equipped SEM's, TEM's and STEM's. Energy loss spectroscopy: In this method, the energies of the inelastically scattered electrons are carefully measured. The energy given up by the primary electron must be equal to that used to eject the electron from an atom of the specimen. Because this process requires differing amounts of energy for each element, the change of the electron's energy is indicative of the atomic composition of the specimen. This method is better suited for elements of low atomic weight than high atomic weight and thus nicely complements x-ray analysis. This technique can be carried out in suitably equipped SEM's, TEM's and STEM's. Other: There are many other analytical methods that we cannot cover here such as analyses using emitted light or auger electrons. These methods are presently not widely used but could become so if technology continues to improve. The scanning tunneling microscope: This is not really a microscope in the sense of the word that we have been using. Basically this type of "microscope" involves positing a stylus with a pointed tip (optimally one that tapers to a tip a single atom wide) at a constant but very small distance (about 1 nm) from a surface (slide 6-10). The distance from the stylus to the surface is small enough to allow electrons to tunnel (that is to go from point a to point b without ever being in the intervening space). This portion of the instrument is almost absurdly small and simple looking (slides 6-11, 6-12). The trick is that it is possible to measure the position of the stylus with atomic precision and thus to build up a computer generated relief map of the surface by measuring the stylus position over the various portions of the
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specimen. This instrument, as befits a microscope invented by IBM, is horrendously complex, and depends on complicated mechanical and electrical components. Such microscopes are capable of imaging individual atoms. Recently, it was used to show that benzene is really a ring structure, and has been used to show the structure of DNA as well (slides 6-12, 6-13). These machines are difficult to use and apparently require much expertise to know if it the microscope is giving an image of your sample or a contaminant. In addition, they can only be used with samples that have a certain level of electrical conductivity. Never-the-less the microscopes have such enormous potential, that the designers have been awarded a Nobel Prize. C. The Atomic Force Microscope: This is a close first cousin of the STM. Here the stylus is actually dragged over the specimen. Through the miracles of physics, the machine can be set up so that enough force can be applied to the stylus that it follows the contours of atoms whilst still not damaging the molecule is it being dragged over. Because the microscope does not use the tunneling current, it can be used to study non-conductive samples. In the first examples of this machine the position of the stylus was determined by adding a metal portion to the probe and measuring the position of the probe with a scanning tunneling microscope. Most modern versions use other mechanisms such as a mirror which reflects light from a laser into an optical detector (slide 6-15). This technique can resolve atoms or small groups of atoms (slide 6-16). In addition, it has been used to micromanipulate small organic molecules (Nature 331:324), a truly amazing accomplishment. D. The near field scanning optical microscope (NSOM): We have previously stated that the laws of optics limit the resolution to about 1/2 wavelength of light (or slightly better using the confocal microscope). Among other things, this results because the lens is several wavelengths removed from the sample. However, by scanning a light source (or detector) very close to the specimen, an image can be generated at a resolution dependent on only the probe size and the probe to sample distance. Thus, the near field optical microscope is sort of a cross between an optical microscope and a scanning tunneling electron microscope. Initial efforts placed a moving hole next to the specimen (slide 6-17). The resolution achieved can never be much better than the size of the hole. Thus increased resolution requires a whole that is only a fraction of a wavelength wide. Unfortunately such holes pass very little light and cannot be easily used for microscopy with visible light. Instead, most modern versions use some sort of fiber probe which concentrates (or generates) light at the end of it (slide 6-18). It is also necessary to have the specimen very close to the probe - Best results are achieved with the probe contacting the sample or within a few nm. (slide 6-20; a=near contact, b=5nm, c=10nm, d=25nm, e=100nm, f=400nm). This is only about the diameter of a ribosome, so it may not be possible to use this technique to study components deep inside of intact cells or organisms. Resolution is up to 20x better than lensed optical microscope, with further improvements expected. It is usually not as good as electron microscopes (slide 6-19). Multiple contrasting methods can be used with the NSOM as they can with the conventional light microscope. E. The X-ray microscope: X-rays have short wavelengths and good penetrating power and would therefore seem to be an ideal energy source for microscopy. However, no one has figured out how to make an good X-ray lens. Non-the-less, it is possible to produce a very small source of X- rays and that source can be allowed to pass through the specimen and the image projected on the screen (in principle just as the slide projector produced a magnified image of the slide
on the screen). Such instruments have a better resolving power than the light microscope but not nearly as good as the EM. Most recent work on this microscope involves focusing X-ray beams with curved X-ray mirrors. These mirrors seriously suffer from aberrations including astigmatism, and result in limited resolution gains. Recently a novel X-ray converging lens has been developed that uses multiple fiber optics as light pipes (actually X-ray pipes). Such lenses are relatively crude (as lenses) and very expensive to make, but could eventually be used to make a microscope. Periodically, you hear about a breakthrough, but the X-ray microscope has a long way to go before it is really useful. However one of the most widely touted components of the "Star Wars" program involves focusing X-rays; perhaps the intense research will result in improvements in this type of microscope. F. The acoustic microscope: All you need for a microscope is some medium with waves and some way to focus these waves. It is possible to build microscopes that use sound waves to study cells. Such microscopes show promise but have not yielded much new information as yet. G. The proton microscope: In principle, you could construct a microscope that used protons instead of electrons. Such microscopes have been made and show promise. One advantage is that the proton has much more penetrating power than the electron and thus could be used with much thicker samples. It would also have some advantages in analytical microscopy. One disadvantage is that the proton source is a small accelerator; I personally would not like to have one of these in my basement.
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378 Lect Notes 13

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378 Lect Notes 13

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Light, Optics and the Microscope

The Brightfield Microscope

Important Optical Characteristics of the Light Microscope

Recording the image

Introduction to Electron Microscopy

The Electron Microscope

The Scanning Electron Microscope

Other Forms of Microscopy

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